Recently, DNA sequences of various organisms including human have been analyzed increasingly and rapidly, and with regard to a large number of genes extracted from this enormous genome sequence information, a research called “Structural genomics” became an important field, which is a systematic and comprehensive analysis of the relationship between structure and function of proteins by determining three dimensional structure of proteins encoded by individual genes. In addition, a high-throughput analytical means applicable to a large scale analysis for promoting such a research is desired.
In this “Structural genomics”, a most important target as a pharmaceutical development among the proteins for their structural analysis may for example be a membrane protein. While a membrane protein is responsible for important cellular functions such as a response to a stimulation, cellular skeleton and adhesion, material transportation and electron transport, it is difficult to be analyzed biochemically because of an extreme difficulty in its isolation and purification.
When a membrane protein is expressed in a cultured cell, it is accumulated in the cell membrane as a result of a localization function of the host cell. Thus, in a purification step of the resultant protein, it is necessary to extract the membrane protein from the cell membrane using various solubilizing agents, which is time-consuming and laborious and also involves a problem with regard to the extraction efficiency. Moreover, some types of the solubilizing agents may deteriorate the structure or function associated naturally with the protein.
On the other hand, a membrane protein, when expressed in E. coli, frequently precipitates as insolubles, which leads to a purification requiring a precipitation solubilizing step employing a potent denaturating agent such as guanidine or urea and a step for recovering the native structure (folding) of the protein which has once been denatured in the preceding solubilization step. These steps are problematic not only in view of the time and the labor but also in view of re-insolubilization occurring during the above-mentioned folding step.
In order to avoid these problems, a prior art employed a method for co-expressing an E. coli heat shock chaperone or foldase with a heterologous gene. For example, JP-A-9-107954 discloses, as this heat shock chaperone, heat shock proteins such as E. coli GroES and GroEL, which serves to catalyze a correct folding of a polypeptide which has been newly synthesized in E. coli. On the other hand, a foldase such as a protein disulfide isomerase or peptidylproryl cis-trans isomerase is disclosed, and an over-co-expression of these chaperone and foldases, especially an E. coli thioredoxin, allows a mouse c-Myb or human transcription factor cAMP-responding element-binding protein or p53 anticancer gene product to be expressed as a soluble protein at a level of several ten mg to 100 mg per 1 liter of an E. coli culture.
Still another method is a method for allowing an intended heterologous gene to be expressed as a fusion protein with another protein. This another protein may for example be glutathione S-transferase (GST), maltose binding protein (MBP), protein A or protein G, and a target protein is expressed usually as a protein fused with the C terminal any of these proteins (see JP-A-9-107954, supra).
However, any of these methods is still problematic because it allows a highly hydrophobic protein such as a membrane protein to be aggregated readily in E. coli cells upon expression and also because it enables the expression only at an extremely low level.
Under the circumstance discussed above, an objective of the invention is to provide a method for producing a highly hydrophobic protein such as a membrane protein in a state allowing the solubilization to be accomplished very easily and also in a state allowing a biologically active three dimensional structure to be formed.